Project Summary/Abstract The overall goal of this application is to validate Lysophosphatidic Acid Acyltransferase-beta (LPAAT-) as a contributor to tumorigenesis and to identify small molecule inhibitors of LPAAT- as novel antitumor agents for the treatment of cancer. LPAAT- is a critical enzyme that converts Lysophosphatidic Acid (LPA) to Phosphatidic Acid (PA), a cofactor needed for the full activation of several signaling proteins such as c-Raf, BRAF, PKC-zeta and mTOR that play pivotal roles in malignant transformation and tumor survival. Furthermore, the LPAAT- generated PA has been implicated in the regulation of proliferation and survival of cancer cells. We have shown in preliminary data that LPAAT is upregulated in various cancers. We have shown that LPAAT-b can enhance Ras-mediated transformation. We have also shown that first generation LPAAT inhibitors inhibit tumor cell proliferation in vitro and tumor growth in vivo. Our hypothesis is that: LPAAT- regulates the growth and survival of human cancer cells and that inhibition of this enzyme is an effective therapeutic strategy. To test this hypothesis we will pursue 3 Aims. In Aim 1 we will determine if LPAAT- possesses independent transforming activity and/or can collaborate with other onco-proteins in enhancing malignant transformation. Our approach will be to construct mammalian expression vectors, to over-express LPAAT- in untransformed immortalized cell lines, and to test the oncogenic potential of LPAAT- in transformation assays in vitro and tumorigenic assays in immunodeficient mice in vivo. In Aim 2, we will determine whether LPAAT- is required for, and/or cooperates with, RalGDS/RalA/B, Raf-1/Mek1/2 or PI3K/Akt pathways to transform immortalized human primary epithelial cells. Two approaches will be used in this aim, one using Ras effector loop mutants that selectively activate each of the 3 pathways in the absence or presence of LPAAT- siRNA. The other approach will directly express Raf-1, PI3K and Ral and determine which of these effectors can be blocked with an LPAAT- siRNA. In Aim 3, we will use high throughput screening and chemistry lead optimization to identify potent and selective LPAAT- inhibitors as novel anticancer drugs. The proposed studies will further validate LPAAT- as a target and identify inhibitors of this enzyme as novel anticancer drugs. Therefore, this proposal advances the mission of the agency to translate our understanding of signaling molecules into new therapies.